Project Summary The past decade has witnessed explosive advances in our understanding of how the organization of chromatin controls gene expression in eukaryotes. Much of the work delineating these mechanisms has contributed to the notion that a so-called ?Histone Code,? which refers to the landscape of histone post- translational modifications (PTMs), is a central determinant of a gene's potential to be activated or repressed in response to environmental stimuli. However, although detected in vivo, very little is known about how non-enzymatic covalent modifications (NECMs), such as glycation, affect the established cellular transcriptional program. We recently found that glycation, which is the hallmark of diabetes, accumulates on histones in a disease state-dependent manner (Zheng et al. Nature Communications, 2019). Using a variety of biophysical, biochemical and genetic methods we found that histone glycation disrupts regulatory histone PTMs as well as changes chromatin architecture in vitro and in cells by forming both histone-histone and histone-DNA crosslinks. Importantly, we identified a cellular regulatory response to this damage in the form of the deglycase DJ-1 as well as more recently, an arginine-specific deglycase, PAD4, which further demonstrates the crosstalk between histone glycation and other enzymatic PTMs (Zheng et al., in revision). In this proposal, we will take an interdisciplinary approach and leverage new chemical probes we developed to determine the sites of glycation on histones and their distribution within chromatin. In addition, we will perform a high-resolution analysis to identify the precise mechanistic effect histone glycation has on higher-order chromatin structure and the epigenetic landscape. Finally, we will investigate the cellular response to glycation by identifying ?readers? and ?erasers? of this new mark. Successful completion of this project is expected to yield a detailed molecular mechanism linking a new class of histone modifications to transcription regulation, thus providing essential insights into a fundamental biological problem and opening the door to new therapeutic avenues.